Repertoire analysis

Clonal abundance, diversity and V-family gene usage

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Bcellmagic analysis pipeline

Pipeline overview

Number of sequences

Number of reads for each of the samples and number of sequences left after representative analysis steps.

ID	Source	Treatment	Extraction_time	Population	Sequences_R1	Sequences_R2	Filtered_quality_R1	Filtered_quality_R2	Mask_primers_R1	Mask_primers_R2	Paired	Build_consensus	Assemble_pairs	Unique	Representative_2	Igblast
QMKMK229AC	CLAD1	Cladribin	baseline	DN	1001901	1001901	998230	929323	950964	889152	874759	523734	90354	59291	8472	6790
QMKMK230AF	CLAD1	Cladribin	baseline	N	601885	601885	599360	566181	571750	543140	533806	402067	97531	92697	6385	4471
QMKMK231AN	CLAD1	Cladribin	baseline	M	1036290	1036290	1033363	964197	980082	919636	904197	780525	135765	124169	4958	4670
QMKMK232AV	CLAD1	Cladribin	baseline	P	1352414	1352414	1346800	1262235	1290104	1212304	1196447	939220	149863	130713	7714	6760
QMKMK233A5	CLAD1	Cladribin	6months	DN	1223690	1223690	1218476	1135468	1160160	1085218	1067347	791259	164348	121167	12720	11196
QMKMK234AD	CLAD1	Cladribin	6months	N	577697	577697	574852	539847	539088	508859	500228	385783	89643	88089	4824	3042
QMKMK235AL	CLAD1	Cladribin	6months	M	801072	801072	799337	750714	761523	718534	706606	608542	111207	95243	4519	4087
QMKMK236AT	CLAD1	Cladribin	6months	P	2043123	2043123	2033938	1877437	1930800	1784871	1761332	1235843	181414	160573	15491	13605
QMKMK241AU	CLAD1	Cladribin	12months	DN	1857453	1857453	1847028	1701105	1709731	1569714	1550126	857641	153794	117452	14305	11726
QMKMK242A4	CLAD1	Cladribin	12months	M	1850771	1850771	1846857	1708178	1770918	1632432	1611884	1280996	217155	195288	10505	7909
QMKMK243AC	CLAD1	Cladribin	12months	N	1244736	1244736	1242282	1135476	1178264	1075176	1059637	679941	139275	136730	15410	4897
QMKMK244AK	CLAD1	Cladribin	12months	P	1811754	1811754	1807946	1664568	1739221	1597527	1577659	1079483	254309	209762	21112	17524
QMKMK533AN	CLAD2	Cladribin	baseline	N	299299	299299	298776	284644	278650	263896	259773	136538	42888	21024	3065	2478
QMKMK534AV	CLAD2	Cladribin	baseline	M	911354	911354	909208	856360	853308	802774	789542	619034	99507	87314	6089	5676
QMKMK535A5	CLAD2	Cladribin	baseline	DN	1548122	1548122	1546518	1485086	1455661	1392202	1373849	746018	162419	91821	14291	12022
QMKMK536AD	CLAD2	Cladribin	baseline	P	828075	828075	826764	781959	769833	729669	715212	583392	116702	101158	7315	7063
QMKMK537AL	CLAD2	Cladribin	6months	N	1695948	1695948	1695010	1636672	1564060	1506398	1482971	541495	162342	145708	25035	10021
QMKMK538AT	CLAD2	Cladribin	6months	M	2467900	2467900	2466808	2356982	2309769	2200497	2169463	767599	187812	126230	19934	17409
QMKMK539A3	CLAD2	Cladribin	6months	DN	1670407	1670407	1669498	1608729	1548656	1486886	1464870	472354	119636	57322	9889	8396
QMKMK540A6	CLAD2	Cladribin	6months	P	1418658	1418658	1417830	1366294	1320682	1272534	1251506	771396	206754	144573	18117	16226
QMKMK541AE	CLAD2	Cladribin	12months	N	1254799	1254799	1253722	1212466	1174117	1107772	1090535	522116	180331	174806	16654	7429
QMKMK542AM	CLAD2	Cladribin	12months	M	2480119	2480119	2478403	2372338	2315848	2199929	2166193	1126337	265733	232685	15947	13290
QMKMK543AU	CLAD2	Cladribin	12months	DN	1492340	1492340	1491521	1430650	1373414	1309222	1288903	743614	187692	140124	15720	12862
QMKMK544A4	CLAD2	Cladribin	12months	P	1104002	1104002	1103382	1063497	1036043	994570	979051	493550	147756	93526	13523	11414
QMKMK545AC	CLAD3	Cladribin	baseline	N	1571620	1571620	1570771	1523584	1497824	1451341	1431781	543127	171669	143577	26458	14092
QMKMK546AK	CLAD3	Cladribin	baseline	M	1798031	1798031	1796794	1739586	1712984	1656005	1636655	1007055	254908	229383	13572	12353
QMKMK547AS	CLAD3	Cladribin	baseline	DN	1325826	1325826	1324051	1277183	1262297	1218805	1199399	629789	146170	105948	13939	11562
QMKMK548A2	CLAD3	Cladribin	baseline	P	1375460	1375460	1374152	1327752	1308934	1264258	1245115	730531	161034	141145	13243	12400
QMKMK549AA	CLAD3	Cladribin	6months	N	1385899	1385899	1385012	1335147	1305595	1255991	1239376	490511	158561	140713	30701	13136
QMKMK550AD	CLAD3	Cladribin	6months	M	1240876	1240876	1240142	1207393	1183864	1149887	1136654	681735	204620	143599	17407	15520
QMKMK551AL	CLAD3	Cladribin	6months	DN	261349	261349	260058	244972	215326	231145	197023	176276	32325	19157	2454	1429
QMKMK552AT	CLAD3	Cladribin	6months	P	235034	235034	234857	229626	224030	219497	216216	172573	77444	53394	7446	7258
QMKMK553A3	CLAD3	Cladribin	12months	N	1591756	1591756	1590676	1540766	1517741	1464383	1445065	515307	173696	149077	28654	15266
QMKMK554AB	CLAD3	Cladribin	12months	M	1564019	1564019	1562831	1507023	1492048	1428178	1411785	779001	213213	166219	19733	15266
QMKMK555AJ	CLAD3	Cladribin	12months	DN	111483	111483	111312	107140	103242	100959	97245	85541	42105	30219	5062	4850
QMKMK556AR	CLAD3	Cladribin	12months	P	155013	155013	154892	151406	146876	144291	141167	87747	48027	25315	4504	4231
QMKMK557A1	CLAD4	Cladribin	baseline	N	1398965	1398965	1392433	1294121	1330529	1238996	1220960	745812	129060	123160	9747	4513
QMKMK558A9	CLAD4	Cladribin	baseline	M	1374928	1374928	1368571	1237182	1305056	1178785	1164891	979676	141173	122743	6397	5864
QMKMK559AH	CLAD4	Cladribin	baseline	DN	2409179	2409179	2398262	2172405	2301729	2083291	2058484	1124304	196878	154809	19618	16839
QMKMK560AK	CLAD4	Cladribin	baseline	P	1068173	1068173	1064298	978142	1021878	940132	928637	709473	170030	146347	9104	8545
QMKMK561AS	CLAD4	Cladribin	6months	N	1641107	1641107	1576042	1345005	1477248	1274519	1229456	874314	68880	67173	2444	132
QMKMK562A2	CLAD4	Cladribin	6months	M	1385399	1385399	1379705	1250203	1320361	1196503	1179563	962386	157538	143239	7102	6064
QMKMK563AA	CLAD4	Cladribin	6months	DN	2589345	2589345	2576384	2318103	2466634	2218225	2186658	1244923	195806	163852	20595	16888
QMKMK564AI	CLAD4	Cladribin	6months	P	1801823	1801823	1794104	1626149	1707738	1549822	1527755	970293	139581	117108	10526	9214
QMKMK565AQ	CLAD4	Cladribin	12months	N	1233912	1233912	1227233	1127439	1171418	1074382	1058954	706032	124861	119933	8175	4157
QMKMK566A0	CLAD4	Cladribin	12months	M	1519136	1519136	1511801	1377911	1458300	1325813	1307238	1038900	165094	139607	10053	9030
QMKMK567A8	CLAD4	Cladribin	12months	DN	1084835	1084835	1077309	963006	985191	874572	862952	641429	113514	106906	8242	4475
QMKMK568AG	CLAD4	Cladribin	12months	P	1322433	1322433	1317876	1210528	1271272	1164834	1150760	665756	151153	105442	9341	7208
QMKMK569AO	CLAD5	Cladribin	baseline	N	1447197	1447197	1445335	1373651	1337298	1269719	1252608	619522	174482	158367	18757	6710
QMKMK570AR	CLAD5	Cladribin	baseline	M	1468661	1468661	1466765	1367672	1319151	1225178	1209652	893529	189690	167978	7970	6680
QMKMK571A1	CLAD5	Cladribin	baseline	DN	1126741	1126741	1125031	1062795	1031127	971370	960077	512019	112116	70502	9251	6405
QMKMK572A9	CLAD5	Cladribin	baseline	P	1397460	1397460	1396014	1316108	1290285	1214297	1199625	843530	178333	134207	12210	10853
QMKMK573AH	CLAD5	Cladribin	6months	N	1329042	1329042	1326871	1258667	1193608	1129648	1114054	535460	159471	147244	17033	5721
QMKMK574AP	CLAD5	Cladribin	6months	M	1237833	1237833	1236499	1162554	1137697	1066878	1053987	813652	164645	137354	6905	6396
QMKMK575AX	CLAD5	Cladribin	6months	DN	1383178	1383178	1381643	1296415	1266158	1181512	1167639	852502	190750	173985	7869	5721
QMKMK576A7	CLAD5	Cladribin	6months	P	1262705	1262705	1261244	1179067	1143706	1064972	1051693	793719	206575	171238	10789	9636
QMKMK577AF	CLAD5	Cladribin	12months	N	1351967	1351967	1350771	1280959	1243575	1172545	1156655	527191	156413	144948	15828	5310
QMKMK578AN	CLAD5	Cladribin	12months	M	713600	713600	712818	676328	667632	628686	622159	517446	126346	101975	4161	3828
QMKMK579AV	CLAD5	Cladribin	12months	DN	2456014	2456014	2451084	2269903	2141182	1958202	1938322	660015	132912	86965	14241	11745
QMKMK580A0	CLAD5	Cladribin	12months	P	834969	834969	834181	787283	779041	731399	722664	580598	147105	115414	6571	6142
QMKMK581A8	CLAD6	Cladribin	baseline	N	1001206	1001206	1000860	970356	962940	936587	920818	443222	140232	86145	18839	12881
QMKMK582AG	CLAD6	Cladribin	baseline	M	1283684	1283684	1283183	1244329	1236314	1203407	1184237	788065	169359	149750	15320	14342
QMKMK583AO	CLAD6	Cladribin	baseline	DN	1097528	1097528	1096978	1066751	1063618	1033309	1021656	634966	162233	105871	17897	16466
QMKMK584AW	CLAD6	Cladribin	baseline	P	1016627	1016627	1016172	988801	984990	962146	947239	442682	163962	76800	20099	18856
QMKMK585A6	CLAD6	Cladribin	6months	N	1285504	1285504	1285176	1251705	1229806	1201931	1182778	543799	171992	148549	28207	18306
QMKMK586AE	CLAD6	Cladribin	6months	M	1330009	1330009	1329416	1292399	1284875	1253526	1233148	891220	225136	195466	18430	16976
QMKMK587AM	CLAD6	Cladribin	6months	DN	1242825	1242825	1240467	1197808	1200344	1156147	1137148	654641	192546	126256	26229	24526
QMKMK588AU	CLAD6	Cladribin	6months	P	1002045	1002045	1001430	967754	965269	932794	921632	434169	147302	84076	18894	16986
QMKMK589A4	CLAD6	Cladribin	12months	N	1137999	1137999	1137344	1103323	1097509	1066778	1049119	462125	140904	135519	21100	13153
QMKMK590A7	CLAD6	Cladribin	12months	M	1209002	1209002	1208461	1171649	1172398	1134117	1117971	734224	260959	216292	24198	22100
QMKMK591AF	CLAD6	Cladribin	12months	DN	1197736	1197736	1197184	1151818	1136481	1087259	1076116	597057	230367	158141	28591	25804
QMKMK592AN	CLAD6	Cladribin	12months	P	992655	992655	992309	965445	964234	935022	924219	613740	189846	142598	19691	18074
QMKMK593AV	CLAD7	Cladribin	baseline	N	1072973	1072973	1072013	1039366	988048	953478	940620	440561	133368	110804	16445	7779
QMKMK594A5	CLAD7	Cladribin	baseline	M	1840517	1840517	1838589	1774842	1701120	1635963	1614648	951547	227992	188707	14405	12975
QMKMK595AD	CLAD7	Cladribin	baseline	DN	1440796	1440796	1438064	1373169	1336916	1271791	1252865	628764	132142	78579	11112	9216
QMKMK596AL	CLAD7	Cladribin	baseline	P	1677725	1677725	1676137	1618758	1542366	1484679	1464832	759842	228681	135178	19623	17045
QMKMK597AT	CLAD7	Cladribin	6months	N	1239270	1239270	1237713	1198319	1123326	1081563	1066953	497751	144787	123197	13744	7291
QMKMK598A3	CLAD7	Cladribin	6months	M	1298404	1298404	1297197	1252956	1196708	1145838	1133594	718360	193455	162681	15846	12138
QMKMK599AB	CLAD7	Cladribin	6months	DN	1254978	1254978	1253336	1203733	1142735	1087229	1075266	641337	171226	151010	14466	9894
QMKMK600AI	CLAD7	Cladribin	6months	P	1479917	1479917	1470919	1412765	1358861	1304396	1281664	714210	66255	62209	3083	1415
QMKMK601AQ	CLAD7	Cladribin	12months	N	1149545	1149545	1148532	1113085	1063607	1023791	1009898	423783	141213	121701	15720	8170
QMKMK602A0	CLAD7	Cladribin	12months	M	1939178	1939178	1936601	1866139	1800331	1717691	1698002	1106596	245522	189688	17128	15293
QMKMK603A8	CLAD7	Cladribin	12months	DN	1545739	1545739	1541982	1470482	1406198	1325331	1310600	517285	81955	48826	7514	6121
QMKMK604AG	CLAD7	Cladribin	12months	P	1211943	1211943	1210523	1172308	1132847	1087549	1075259	486518	138915	90522	13011	10486
QMKMK605AO	CLAD8	Cladribin	baseline	N	1444889	1444889	1444445	1385795	1385248	1321867	1304667	533333	155798	139077	20017	6759
QMKMK606AW	CLAD8	Cladribin	baseline	M	1703561	1703561	1701051	1643939	1631017	1568457	1549559	1091551	151421	140102	8872	5779
QMKMK607A6	CLAD8	Cladribin	baseline	DN	1108324	1108324	1107613	1067244	1061080	1015816	1004178	528412	115795	81992	8232	6779
QMKMK608AE	CLAD8	Cladribin	baseline	P	1540452	1540452	1539639	1477308	1482318	1412976	1397948	991926	207541	162117	13883	12397
QMKMK609AM	CLAD8	Cladribin	6months	N	834333	834333	834049	798958	780110	742394	733064	418241	125716	105779	11088	5615
QMKMK610AP	CLAD8	Cladribin	6months	M	1106235	1106235	1104644	1054961	1057632	1004099	991576	776744	157852	130084	6879	6471
QMKMK611AX	CLAD8	Cladribin	6months	DN	1303870	1303870	1303135	1240914	1243559	1172185	1160462	730474	200257	157489	15954	11884
QMKMK612A7	CLAD8	Cladribin	6months	P	968338	968338	967925	925579	923220	878144	867982	661725	189758	152493	10350	8882
QMKMK613AF	CLAD8	Cladribin	12months	N	1798683	1798683	1798081	1723011	1727265	1641478	1620069	633213	173676	154775	21448	8049
QMKMK614AN	CLAD8	Cladribin	12months	M	1186393	1186393	1185783	1139636	1142249	1085053	1073036	752118	179324	153196	8280	7679
QMKMK615AV	CLAD8	Cladribin	12months	DN	1407945	1407945	1407291	1342821	1314510	1238663	1225435	715687	188862	157705	12270	9190
QMKMK616A5	CLAD8	Cladribin	12months	P	1029639	1029639	1029226	988857	995363	947316	936477	661648	182340	158080	7835	7172

Plotting number of unique sequences

Plotting number of representative 2 sequences

Plotting number of Igblast identified sequences

Clonal abundance

For plotting the clonal abundance, the clones were ordered by size from bigger clones to smaller clones (x-axis, Rank). The Abundance of each clone was represented as the percentage of unique sequences in the clone, with respect to the total number of unique sequences in that subject (By Patient) or in the B-cell or T-cell sample (By Cell Population).

To correct for the different number of sequences in each of the samples, the Bootstrapping technique was employed, in which 200 random bootstrap samples were taken, with size the number of sequences in the sample with less sequences (N). The solid line shows the mean Abundance of the bootstrap samples, whereas the transparent area shows the full Abundance range of the bootstrap samples.

All clonal abundance plots and tables with abundance values can be found under repertoire_analysis/Abundance.

Clonal abundance per subject

Calculate area under the curve for abundance

Count clones per subject

Clonal abundance per cell population

If different types of B-cell or T-cell populations are provided, here the clonal abundance is plotted for each patient and B / T-cell population.

Clonal diversity

The clonal diversity \(D\) of the repertoire was calculated according to the general formula of Hill Diversity numbers:

\[ \begin{aligned} ^{q}D = \left( \sum_{i=1}^Rp_i^q \right)^{1/(1-q)} \end{aligned} \]

where:

• \(p_i\) is the proportion of unique sequences belonging to clone \(i\).
• \(q\) are the values of the different diversity numbers.
• \(R\) is the Richness, the number of different clones in the sample.

At \(q=1\) the function is undefined and the limit to zero equals the exponential of the Shannon Entropy:

\[ \begin{aligned} ^{1}D = exp \left( \sum_{i=1}^Rp_i ln(p_i) \right) \end{aligned} \]

The intuition about the different Hill Diversity values is the following:

• At \(q=0\) the diversity index equals the number of clones in the sample.
• At \(q=1\) the diversity index is the geometric mean of the clones in the sample, weighted by their proportion in the sample.
• At \(q>1\) more weight is given to the clones with higher proportions in the sample.

All clonal diversity plots and tables with diversity values can be found under repertoire_analysis/Diversity. To correct for the different number of sequences in each of the samples, the Bootstrapping technique was employed, in which 200 random bootstrap samples were taken, with size the number of sequences in the sample with less sequences (N). The solid line shows the mean Diversity of the bootstrap samples, whereas the transparent area shows the full Diversity range of the bootstrap samples.

Clonal diversity per subject

Clonal diversity at specific q values

q=1

q=0

Clonal diversity per cell population

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Clonal diversity per population at specific q values

q=1

q=0

V gene usage

V gene family usage

The V gene usage (in percentage) in each of the samples is represented below. All plots and tables can be found here.

Gene family usage is normalized by the number of clones.

By patient

By Population

V gene usage

The V gene usage (in percentage) in each of the samples is represented below. All plots and tables can be found here.

By clones

By sequences

Isotype usage

Isotype usage per subject

`summarise()` has grouped output by 'isotype', 'sample', 'source', 'treatment'. You can override using the `.groups` argument.

Isotype usage per cell population

`summarise()` has grouped output by 'isotype', 'sample_pop', 'source', 'treatment', 'extract_time'. You can override using the `.groups` argument.

Clonal overlap analysis

'repertoire_comparison/Clone_overlap' already existsError in paste(outdir, "Clone_overlap", names(df_pat)[i], sep = "/") : 
  object 'i' not found

Citations

If you use nf-core/bcellmagic for your analysis, please cite it using the following DOI: 10.5281/zenodo.3607408

Please also cite the nf-core publication (P. A. Ewels et al. 2020).

In addition, citations for the tools and data used in this pipeline are as follows:

• pRESTO (Vander Heiden et al. 2014)
• SHazaM, Change-O (Gupta et al. 2015)
• Alakazam (Stern et al. 2014)
• TIgGER (Gadala-Maria et al. 2015)
• FastQC (Andrews et al. 2010)
• MultiQC (P. Ewels et al. 2016)

Andrews, Simon et al. 2010. “FastQC: A Quality Control Tool for High Throughput Sequence Data.”

Ewels, Philip A., Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso, and Sven Nahnsen. 2020. “The Nf-Core Framework for Community-Curated Bioinformatics Pipelines.” Nature Biotechnology 38 (3): 276–78. https://doi.org/10.1038/s41587-020-0439-x.

Ewels, Philip, Måns Magnusson, Sverker Lundin, and Max Käller. 2016. “MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report.” Bioinformatics 32 (19): 3047–48.

Gadala-Maria, Daniel, Gur Yaari, Mohamed Uduman, and Steven H. Kleinstein. 2015. “Automated Analysis of High-Throughput b-Cell Sequencing Data Reveals a High Frequency of Novel Immunoglobulin v Gene Segment Alleles.” Proceedings of the National Academy of Sciences of the United States of America 112 (8): E862–870. https://doi.org/10.1073/pnas.1417683112.

Gupta, Namita T., Jason A. Vander Heiden, Mohamed Uduman, Daniel Gadala-Maria, Gur Yaari, and Steven H. Kleinstein. 2015. “Change-o: A Toolkit for Analyzing Large-Scale b Cell Immunoglobulin Repertoire Sequencing Data.” Bioinformatics 31 (20): 3356–58. https://doi.org/10.1093/bioinformatics/btv359.

Stern, Joel N. H., Gur Yaari, Jason A. Vander Heiden, George Church, William F. Donahue, Rogier Q. Hintzen, Anita J. Huttner, et al. 2014. “B Cells Populating the Multiple Sclerosis Brain Mature in the Draining Cervical Lymph Nodes.” Science Translational Medicine 6 (248). https://doi.org/10.1126/scitranslmed.3008879.

Vander Heiden, Jason A., Gur Yaari, Mohamed Uduman, Joel N. H. Stern, Kevin C. O’Connor, David A. Hafler, Francois Vigneault, and Steven H. Kleinstein. 2014. “pRESTO: A Toolkit for Processing High-Throughput Sequencing Raw Reads of Lymphocyte Receptor Repertoires.” Bioinformatics 30 (13): 1930–32. https://doi.org/10.1093/bioinformatics/btu138.